EP2687224A1 - Medikament zur Wundbehandlung - Google Patents

Medikament zur Wundbehandlung Download PDF

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Publication number
EP2687224A1
EP2687224A1 EP12177127.3A EP12177127A EP2687224A1 EP 2687224 A1 EP2687224 A1 EP 2687224A1 EP 12177127 A EP12177127 A EP 12177127A EP 2687224 A1 EP2687224 A1 EP 2687224A1
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European Patent Office
Prior art keywords
stathmin
cells
wound
cell
stimulating
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EP12177127.3A
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English (en)
French (fr)
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Rudolf Berger
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Tissue Med Biosciences Forschungs- und Entwicklungsgesellschaft mbH
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Tissue Med Biosciences Forschungs- und Entwicklungsgesellschaft mbH
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Priority to EP12177127.3A priority Critical patent/EP2687224A1/de
Priority to PCT/EP2013/065215 priority patent/WO2014013027A1/en
Priority to CN201380048505.9A priority patent/CN104755095A/zh
Priority to AU2013291972A priority patent/AU2013291972A1/en
Priority to CA2879472A priority patent/CA2879472A1/en
Priority to US14/415,658 priority patent/US20150202258A1/en
Priority to EP13739976.2A priority patent/EP2874644A1/de
Priority to JP2015522102A priority patent/JP2015524402A/ja
Publication of EP2687224A1 publication Critical patent/EP2687224A1/de
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0066—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to the field of medicaments for regenerative treatments, in particular wound healing.
  • Wound healing constitutes a complex, dynamic, and well-orchestrated process that is activated whenever disruption of the skin tissue occurs.
  • a wide spectrum of events takes place through the wound-healing process including platelet aggregation, coagulation cascade activation, cell migration, cell proliferation, inflammatory infiltration, cellular differentiation, and tissue remodeling.
  • the cascade of these events seems to be well discriminated and divided into three phases, namely inflammation, proliferation, and wound contraction and remodeling, they actually overlap with each other and continued or renewed tissue damage may re-initiate the sequence at affected sites within an existing wound.
  • Impaired wound healing is associated with increased morbidity and mortality. Non-healing wounds often lead to amputation. However, despite an extensive research, the exact underlying pathogenetic mechanisms have not been fully elucidated. It appears to be the net result of micro- and macrovascular disease. Wound healing is typically delayed in diabetes mellitus and angiopathy seems to contribute significantly. In addition, neuropathy and the lack of growth factors supporting mesenchymal cell migration and activation has been recognized as one of the causes for delayed diabetic wound healing. Other factors that appear to be innate to the diabetic state itself, such as changes in cell morphology and function, have been implicated in its pathogenesis.
  • WO 2007/089151 A1 identified stathmin as TLR3 activating substance and suggested the treatment of degenerative inflammatory processes in neurodegenerative disorders such as Alzheimer's disease. Also the treatment of wounds is mentioned.
  • US 6,429,304 B1 teaches compounds with anti-mitotic activity that can be used to treat various proliferative diseases such as cancer or "abnormal wound healing".
  • the mode of action is a prevention of cell growth.
  • the anti-mitotic activity of the compounds is based on an alleged microtubuli depolymerization ability.
  • One such compound is stathmin, also referred to as OP18.
  • WO 2004/113561 A2 is a further document stating that stathmin or oncoprotein 18 prevents uncontrolled cell growth due to tubulin decreases.
  • stathmin has any beneficial effect in wound healing.
  • the present invention provides the use of stathmin, a nucleic acid encoding stathmin or a cell expressing stathmin in the treatment of wounds.
  • the present invention also relates to stathmin, a nucleic acid encoding stathmin or a cell expressing stathmin for use in the treatment of wounds.
  • the invention relates to the use of stathmin, a nucleic acid encoding stathmin or a cell expressing stathmin in the preparation of a medicament in the treatment of wounds.
  • pharmaceutical compositions comprising stathmin.
  • the invention further provides the in vitro or in vivo method of increasing or inducing the proliferation and/or migration of mesenchymal cells, epidermal cells and/or stem cells, in particular stromal stem cells, for stimulating immune cells, preferably natural killer cells, for stimulating fibroblasts, for stimulating epithelial cells, preferably epithelial cells of the epidermis, or for stimulating angiogenesis, in particular for stimulating IL-8 production in said cells, especially in mesenchymal cells and/or fibroblasts. All of these aspects are equivalent and all preferred embodiments relate to each one of these aspects equally.
  • stathmin as a very potent wound healing accelerator, that even surpassed common medicaments such as PDGF formulations.
  • common medicaments such as PDGF formulations.
  • stathmin could achieve wound closure at an unexpected fast rate.
  • Stathmin is a protein with known sequence (NCBI database NP_005554 and CAD19057 or SEQ ID NO: 1 (stathmin a) or SEQ ID NO: 2 (stathmin b)).
  • Stathmin and its isoforms are further known from the background art mentioned above. It is an ubiquitously expressed protein found in most tissues in mammals. Many different phosphorylated forms are observed depending on specific combinations among the sites which can be phosphorylated. Phosphorylation at Ser-16 seems to be required for neuron polarization. Phosphorylation at Ser-63 reduces tubulin binding 10-fold and suppresses the MT polymerization inhibition activity.
  • Plasmacytoid dendritic cells have a distinctive role in wounded skin and are involved in the acute inflammatory response and wound healing through their production of growth factors.
  • a human dendritic cell line was developed which secretes a unique set of proteins, which are instrumental for tissue regeneration.
  • stathmin a protein with a molecular weight of 17,3kD (SEQ ID NO: 1).
  • the stathmin gene from pDCs was cloned and expressed in E. coli and purified to homogeneity.
  • stathmin induces proliferation and migration of human mesenchymal cells, supports neoangiogenesis and stimulates natural killer cell activity.
  • the absence of dermal cell proliferation, new vessels and a first line defense against bacterial infections are the key problems in many chronic wounds. Striking results were obtained in vivo when wound healing in Zucker rats which have a delayed wound healing due to a metabolic syndrome was studied.
  • stathmin significantly outperformed PDGF.
  • PE-tomography with 124 I-labelled stathmin showed that the protein remained in the wounded area.
  • Stathmin is remarkable stable in wound fluid (more than 8 hrs) and is not detectable in serum when applied topically, no systemic exposure was measured.
  • the present invention provides for the first time a use of stathmin in the treatment of wounds supported by experimental evidence.
  • the present invention further provides the use of stathmin in non-healing wounds (non-healing without the inventive treatment) or in treating wounds with reduced or impaired wound healing.
  • Wounds with reduced or impaired wound healing are e.g. chronic wounds or wounds with disease- or medication-dependent impaired wound healing (which may also be or develop into chronic wounds).
  • nucleic acids that encode stathmin and lead to expression thereof in a cell or the use of such a cell in such a treatment are also possible.
  • the invention further provides the combination of stathmin with collagen in the treatment of wounds. This combination shows even further increased wound healing rates due to synergistic effects.
  • Stathmin as used herein relates to any stathmin isoform or variant or any post-translationally modified form, including phosphorylated forms.
  • stathmin comprises amino acids 1 to 126 of SEQ ID NO: 1, or an amino acid sequence that has at least 70 % sequence identity with the sequence of amino acids 1 to 126 of SEQ ID NO: 1.
  • the amino acids 1 to 126 of SEQ ID NO: 1 are also found in SEQ ID NO: 2 and appear to be of a homologous region.
  • Further preferred stathmin variants are disclosed in WO 2007/089151 , US 6,429,304 B1 , WO 2004/113561 A2 and NCBI database entries NP_005554 and CAD19057 (all incorporated herein by reference).
  • sequence identity refers to identity between two sequences, usually amino acid sequences, which can be determined by sequence alignment programs like BLAST, PSI-BLAST (www.ncbi.nlm.nih.gov/blast/) or ClustalW (www.ebi.ac.uk/clustalw/). These algorithms calculate the best match for the selected sequences, and line them up so that the identities, similarities and differences can be seen.
  • the inventive stathmin comprises a sequence with at least 75 %, more preferred at least 80 %, at least 85%, at least 90%, at least 95%, at least 98% or even 100%, sequence identity with the sequence of amino acids 1 to 126 of SEQ ID NO: 1.
  • the inventive stathmin comprises a sequence with at least 70 %, more preferred at least 75%, at least 80 %, at least 85%, at least 90%, at least 95%, at least 98% or even 100%, sequence identity with the sequence as set forth in SEQ ID NO: 1.
  • Stathmin may be stathmin-1, stathmin-2, stathmin-3 or stathmin-4, preferably it is stathmin-1.
  • Stathmin can be a recombinant stathmin. Further preferred it is of the same origin as the patient but also stathmin variants from other animals can be used.
  • the patient is a mammal, especially a human or non-human animal, in particular a domestic animal, such as pig, rodents, or a primate.
  • the stathmin is human stathmin or stathmin from a non-human animal, in particular a domestic animal, such as pig, rodents, or a primate.
  • stathmin of the present invention may have further amino acids or protein components. It may be a fusion protein.
  • extended stathmin proteins may have in preferred embodiments a limited size, e.g. up to 2000 amino acids, up to 1800 amino acids, up to 1600 amino acids, up to 1400 amino acids, up to 1200 amino acids, up to 1000 amino acids, up to 800 amino acids, up to 600 amino acids, up to 400 amino acids, up to 300 amino acids, up to 200 amino acids, or up to 160 amino acids.
  • stathmin proteins that consist of any one of said sequences comprised in the above mentioned embodiments. "Consisting" is used in a closed and sequence limiting meaning.
  • stathmin is phosphorylated.
  • Phosphorylation is a natural post-transcriptional process and occurs during recombinant expression.
  • Possible phosphorylation sites are at amino acids corresponding to amino acids 16, 25, 28, 38, 63, 146 of SEQ ID NO: 1.
  • Preferred phosphorylation are at amino acids corresponding to amino acids 16 and/or 63 of SEQ ID NO: 1.
  • the inventive stathmin may comprise 1, 2, 3, 4, 5 or 6 amino acid phosphorylations.
  • stathmin is acetylated.
  • Acetylation is a natural post-transcriptional process and occurs during recombinant expression.
  • Possible acetylation sites are at amino acids corresponding to amino acids 2, 9, 80, 95, 100, 119, 128 of SEQ ID NO: 1.
  • the inventive stathmin may comprise 1, 2, 3, 4, 5, 6 or 7 amino acid acetylations.
  • stathmin can be glycosylated or not glycosylated. E. coli produced stathmin is not glycosylated and still effective.
  • stathmin proteins it is also possible to use nucleic acids that encode the above mentioned stathmin protein.
  • Such a sequence is e.g. set forth in SEQ ID NO: 3, which encodes SEQ ID NO: 1.
  • Nucleic acids can be used to induce production of stathmin in cells near the wound.
  • Preferred formulations for nucleic acid delivery are e.g. liposomes, microemulsions, micelles or vesicles for controlled delivery.
  • the cell may then produce and secrete stathmin to provide a continuous production of the therapeutic agent.
  • a cell that expresses stathmin for the inventive uses preferably continuously secretes stathmin to provide for the therapeutic effect.
  • a cell can be any cell.
  • the cell is a pDC but also non-pDC's are possible.
  • the cell is not-immunogenic to the patient, e.g. it is a cell obtained from the patient that has been genetically engineered to recombinantly express stathmin. This modification of the cell can be performed in vitro or in vivo.
  • stathmin protein the nucleic acid and the cells equally, in particular since the directly active therapeutic agent of the nucleic acid or the cell is also the expressed stathmin.
  • the present invention provides in particular stathmin, a nucleic acid encoding said stathmin or a cell expressing stathmin for use in the treatment of chronic wounds or wounds with defective angiogenesis in a patient.
  • the treated tissue is the skin or the treated tissue or area comprises at least in part the skin.
  • Chronic wounds or wounds with impaired wound healing may also affect subcutaneous tissues, even the bone.
  • the treatment is preferably topical, especially with a topical formulation such as a hydrogel.
  • the nucleic acid or the cell can be formulated with or coupled to a biocompatible matrix, preferably comprising collagen, gelatin, chitosan and/or hyaluronan, for use in the treatment of wounds, preferably chronic wounds or wounds with defective angiogenesis, in a patient.
  • a biocompatible matrix preferably comprising collagen, gelatin, chitosan and/or hyaluronan
  • this formulation provides surprisingly new properties and effects that are novel over any other prior formulation. Therefore the invention also provides the use of stathmin in such a formulation for the treatment of wounds, including acute and chronic wounds and wounds with impaired wound healing.
  • the preparation of biocompatible matrices is well known in the art and e.g. described in Tan et al. (9).
  • Biocompatible matrices preferably support wound healing and/or adhesion of cells, in particular in the wound. They lead to a strengthening of the regrown cellular scar tissue.
  • the biocompatible matrix comprises a scaffold with hy
  • the stathmin is formulated in a hydrogel.
  • Hydrogels preferably comprise gelatin, alginate, agarose, methylcellulose, hyaluronan or any combination thereof.
  • Organo-chemical hydrogels may comprise polyvinyl alcohol, sodium polyacrylate, acrylate polymers and copolymers with an abundance of hydrophilic groups.
  • Hydrogels comprise a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 99.9% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. They provide a reservoir in topical drug delivery.
  • the wound to be treated is a chronic wound.
  • a chronic wound shows no or limited wound healing. It is a wound that does not heal in an orderly set of stages and in a predictable amount of time.
  • the chronic wound is a wound that has no or reduced wound healing (e.g. only about 20 % wound area closure) within 14 days, preferably 18 days, especially preferred 22 days, even more preferred 28 days, within 34 days, 40 days, 50 days or even 60 days.
  • the wound is anoxic and/or lacks sufficient oxygen supply and/or lacks newly formed arteries.
  • the present invention also stimulates angiogenesis and can help to restore proper or even accelerated wound healing as well as angiogenesis, in a wound or elsewhere.
  • the wound is a wound with disease-dependent impaired wound healing (disease-dependent impaired wound healing can also be chronic as defined above).
  • Disease-dependent impaired wound healing can due to a disease selected from diabetes or metabolic syndrome, chronic venous insufficiency (CVI), peripheral artery occlusive disease (PAOD), cancer, autoimmunity, especially an autoimmunity selected from rheumatoid arthritis, lupus (in particular systemic lupus erytematodes) and livedoid vasculopathy, surgical wounds, ostomy, prolonged inflammatory processes, cellular senescence such as in decubitus growth factor deficiency or growth factor receptor deficiency.
  • the patient treated according to the invention may have one or more of these diseases or conditions. Preferably said patient suffers from diabetes or metabolic syndrome.
  • the wound is a wound with medication-dependent impaired wound healing.
  • Medication-dependent impaired wound healing can be due to a medication selected from treatments with a cortiocosteroid, nicotine, an antibiotic, an immunosuppressant, an anti-coagulant, a cytotoxic medication, an anti-rheuma-medication, especially an anti-rheumatoid arthritis medication, a vasoconstrictor.
  • the patient treated according to the invention may have received one or more of these medications.
  • a patient with tissues with insufficient artery supply may be treated.
  • the patient may have anoxic tissues that are treated by the present invention.
  • the tissue with insufficient artery supply may also be chronic, e.g. be in this state for 14 days, preferably 18 days, especially preferred 22 days, even more preferred 28 days, for 34 days, 40 days, 50 days or even 60 days.
  • Chronic diseases may have acute causes, such as surgical or accidental wounds, or chronic causes, such as in diabetes or other comorbidities, especially a combination with other diseases that cause reduced oxygenation of the skin or other tissues.
  • the present invention relates to a treatment of a patient who is in need of an induction of angiogenesis with stathmin, in particular in the tissue in need of such therapy.
  • the therapy is topical (as is preferred for all embodiments of the invention).
  • the patient does not suffer from a systemic degenerative inflammatory process.
  • the treated wound may or may not comprise a degenerative inflammatory process.
  • Further treatments are of patients who are in need of a stimulation of the innate immune response and/or in need of a induction of cytokines/growth factors and/or TIMPs for immune modulation.
  • Such patients may have a locally or systemic immune deficiency.
  • the immune deficiency may be an insufficient supply (locally or systemically) of all immune cells or a limited immune cell activation or a limited supply (locally or systemically) of particular immune cells, especially cells of the innate immune system, such as NK cells, which are preferably stimulated according to the invention.
  • Locally preferably relates to the wounded area.
  • the immune deficiency is a deficiency in cell-mediated immunity and/or deficiency in the innate immune system.
  • the immune system is divided into a more primitive innate immune system, and acquired or adaptive immune system, each of which contains humoral and cellular components.
  • a nucleic acid or cell can be used to treat a wound in a patient who suffers from diabetes or metabolic syndrome.
  • Diabetes and the metabolic syndrome cause a dysregulation in the patients proliferative and metabolic capabilities, which leads to reduced or no wound healing. Further, diabetes causes immune compromise and damage to small blood vessels, preventing adequate oxygenation of tissue, which can cause chronic wounds even without acute causes.
  • wounds or conditions that can be treated with stathmin are wounds selected from diabetic foot wound, especially a diabetic foot ulcer, an ulcer in general, in particular venous ulcer, a decubitus or pressure ulcer, burns, preferably a third degree burn, a surgical wound, an accidental wound, a necrotic wound, an infected wound.
  • wounds and conditions may be chronic but in special cases also acute.
  • chronic versions of all of these wounds and conditions are treated according to the invention.
  • the present invention further relates to a pharmaceutical composition comprising stathmin.
  • Pharmaceutical compositions or formulations for therapeutic use may comprise a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
  • the invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of stathmin.
  • therapeutically effective amount means an amount which provides a therapeutic effect for a specified condition and route of administration.
  • the composition may be in a liquid or lyophilized form and comprises a diluent (Tris, acetate or phosphate buffers, NaCl) having various pH values and ionic strengths, solubilizer such as Tween or Polysorbate, carriers such as human serum albumin or gelatin, preservatives such as thimerosal or benzyl alcohol. Selection of a particular composition will depend upon a number of factors, including the condition being treated, the route of administration and the pharmacokinetic parameters desired.
  • a diluent Tris, acetate or phosphate buffers, NaCl
  • solubilizer such as Tween or Polysorbate
  • carriers such as human serum albumin or gelatin
  • preservatives such as thimerosal or benzyl alcohol.
  • compositions for topical administration are formulations for topical administration.
  • a hydrogel are also encompassed.
  • compositions comprising stathmin modified with water soluble polymers to increase solubility, stability, plasma half-life and bioavailability.
  • Compositions may also comprise incorporation of stathmin into liposomes, microemulsions, micelles, microparticles or vesicles for controlled delivery over an extended period of time.
  • stathmin is provided with a carrier.
  • the carrier is preferably selected from a gel, preferably a hydrogel, or a wound dressing or a swab, optionally impregnated with a solution containing stathmin.
  • Further carriers comprise carriers for slow-release which release the active agent combination as a longer effective application delayed or slower. Such a preparation with a corresponding carrier is especially suitable for topical and quick administration.
  • stathmin is used in amounts of between 0.1 ⁇ g to 1000 ⁇ g stathmin per cm 2 of the wound. Also preferred are amounts of at least 0.1 ⁇ g at least 0.2 ⁇ g, at least 0.5 ⁇ g, at least 1 ⁇ g, at least 2 ⁇ g, at least 5 ⁇ g and/or at most 1000 ⁇ g, at most 750 ⁇ g, at most 500 ⁇ g, at most 400 ⁇ g, at most 300 ⁇ g, at most 200 ⁇ g, at most 100 ⁇ g or at most 50 ⁇ g per cm 2 of the wound or any range in between these values.
  • the pharmaceutical composition may comprise stathmin formulated with or coupled to a biocompatible matrix, preferably comprising collagen, gelatin, chitosan and/or hyaluronan, as described above.
  • the pharmaceutical composition may also comprise a hydrogel with stathmin as described above.
  • the present invention provides the pharmaceutical composition for use as a medicament.
  • Particular uses are in wound healing and/or the stimulation of angiogenesis as described above.
  • the present invention also provides a method of increasing or inducing the proliferation and/or migration of mesenchymal cells, epidermal cells and/or stem cells, in particular stromal stem cells or hematopoietic stem cells, for stimulating immune cells, for stimulating fibroblasts, for stimulating epithelial cells, preferably epithelial cells of the epidermis or of blood vessels, or for stimulating angiogenesis.
  • This method can be performed in vitro, e.g. on isolated cells or cell cultures.
  • the inventive method can be used for stimulating IL-8 production in said cells, especially in mesenchymal cells and/or fibroblasts.
  • the invention stimulates IL-8 production, which has particular beneficial effects on wound healing.
  • this method in vivo, in particular on a patient with a wound or in need of such a treatment, e.g. for stimulating angiogenesis.
  • the method of increasing or inducing the proliferation and/or migration of mesenchymal cells, epidermal cells and/or stem cells, in particular stromal stem cells or hematopoietic stem cells, for stimulating immune cells, for stimulating fibroblasts, for stimulating epithelial cells, preferably epithelial cells of the epidermis or of blood vessels, or for stimulating angiogenesis in a patient comprising the step of administering stathmin, a nucleic acid encoding said stathmin or a cell expressing stathmin to the patient, preferably by topical administration on a wound.
  • the patient may be in need of such increase, induction or stimulation, e.g. due to a wound, or anoxic, necrotic or infected (or a combination thereof) tissue.
  • inventive method can be used for stimulating IL-8 production in said cells, especially in mesenchymal cells and/or fibroblasts as stated above.
  • Immune cells that can be stimulated are preferably cells of the innate immune system, in particular natural killer cells.
  • the present invention further provides the stimulation of any one of the growth factors as given in table 2 below, especially IFN-alpha, IFN-beta, PDGF-B, FGF-2, HGF, TGF-beta, VEGF and/or KGF, or of IL-8.
  • This method can be used in the above described in vitro or in vivo method.
  • SNF Primary cells
  • PS Primary cells
  • AB4 cells are human cells resembling plasmacytoid dendritic cells and are derived from a patient with histiocytosis.
  • ECV304 and A431 are human cell lines of endothelial and epithelial origin, respectively.
  • Cells were cultured in RPMI1640 supplemented with 10% heat-inactivated FCS, 2 mM L-Glutamine and 100 IU/ml Penicillin-100 ⁇ g/ml Streptomycin (all reagents from PAA Laboratories, Linz, Austria). Cells were sub-cultured twice weekly at a ratio of 1:4.
  • Recombinant IL-2, VEGF and PDGF were obtained from PeproTech (London, UK).
  • Bovine Collagen type I and Fibronectin were purchased from Sigma-Aldrich (St. Louis, MO).
  • Hydrogel refers to Normlgel® (Mölnlycke, Gothenburg, Sweden) and the PDGF-containing Hydrogel was obtained through Janssen-Cilag (Regranex®, Belgium).
  • AB4 cDNA was generated with ImProm-II Reverse Transcription System (Promega) according to the supplier's instruction.
  • a PCR reaction was set up with Phusion High Fidelity DNA Polymerase, Oligonucleotides "hu STAT1-NcoI-F” and “hu STAT1-Bsu-R” and AB4-cDNA as template to generate a PCR product with NcoI restriction site at the 5'-end and Bsu36I restriction site at the 3'-end.
  • the PCR product and pTriEx4-Neo plasmid DNA were digested with NcoI and Bsu36I at 37°C for 2 hours. Digestion products were separated by agarose gel electrophoresis. The respective bands for the digested PCR product and plasmid DNA were purified from agarose gel and ligated with T4 DNA Ligase for 4 hours at room temperature. Ligation reaction was transformed into TOP10 One Shot Chemically Competent cells with heat shock at 42°C for 30 sec. Transformation reaction was plated out on LB Agar plates with Ampicillin and incubated for 12 hours at 37°C. Colony PCR was carried out with GoTaq DNA Polymerase and Oligonucleotides "hu STAT1-NcoI-F" and "hu STAT1-Bsu-R" to identify positive transformants.
  • PCR products were checked on an agarose gel, positive transformants showed a specific band at 450 bp in agarose gel.
  • the positive clones were inoculated into 4 ml LB-Broth with Ampicillin, incubated 12 hours at 37°C.
  • Plasmid preparation was carried out with High Pure Plasmid Isolation Kit according to supplier's instruction. Plasmid DNA was digested with NcoI and Bsu36I at 37°C for 2 hours to further verify the positive transformants.
  • hu STAT1 was successfully cloned in pTriEx4-Neo and TOP10 One Shot Chemically Competent cells. After the NcoI/Bsu36I control digestion hu STAT1 in pTriEx4-Neo /TOP10 clones 4, 6 and 8 showed the respective band at 450bp in agarose gel (see Fig.1 ). Plasmid DNA of the positive transformants was sent to MWG Biotech (Munich, Germany) for sequence verification and evaluation was performed by aligning the resulting sequence with reference hu STAT1 sequence from genbank employing multiple sequence alignment tool.
  • BL21 DE3 E. coli clone 4-3 containing hu Stat1 cloned in pTriEX-4 Neo at NcoI/Bsu36I was grown in LB-Medium containing 100 ⁇ g/ml Ampicillin to OD 600 >0.5.
  • the specific protein band for hu Stat1 (17,3 kD) was induced after addition of 1 mM IPTG.
  • Bacterial cells were harvested by centrifugation at 4,500 rpm for 1 hour. Cell pellet was resuspended in lysis buffer (Pellet from 10 ml culture in 500 ⁇ l Lysis Buffer) and exhibited to 3 freeze/thaw cycles to perform cell lysis. Then the lysate was separated into cell debris and supernatant by centrifugation at 4500 rpm for 1 hour. Lysate-supernatant was filtrated through 0.45 ⁇ m syringe filter.
  • the lysate-supernatant was applied to Capto DEAE Sepharose Fast Flow (GE Healthcare, Germany) with Anion Exchange Buffer A on an ⁇ KTA Purifier FPLC System. Unbound sample was washed from the column with 10CV of Anion Exchange Buffer A, then the column was washed with 10CV of 5% Anion Exchange Buffer B. For elution 5CV of 20% Anion Exchange Buffer B were applied to the column. The eluate was collected in fractions of 15 ml and analyzed on 12% BisTris Gel with Coomassie Stain.
  • the hu STAT1 containing fractions from the Anion Exchange Chromatography were pooled and precipitated with 30% Ammoniumsulfate stirring for 24 hours at 4°C. After precipitation the sample was separated in pellet and supernatant by centrifugation at 4500 rpm for 10 minutes. The supernatant was filtered through 0.45 ⁇ m syringe filter.
  • the fractions containing hu STAT1 from the HIC step were pooled and concentrated with a Centrifugal Filter Device (Millipore, Vienna), in this step also buffer was exchanged to 20 mM Tris pH 7.3. Finally the product was sterilized through 0.2 ⁇ m syringe filter. The purity of the resulting product was proven by Silver Staining (see Figure 3 lane 10). Protein identity was verified in a target protein specific Western Blot (see Figure 3 , lane 12).
  • hu STAT1 in pTriEx4 Neo/TOP10 clone 4 is identical with huSTAT1 and correctly ligated into the pTriEx4-Neo vector at the restriction sites 5'-NcoI and 3'-Bsu36I, thus enabling expression of the native protein without vector-encodes amino acids (see Figure 2 ).
  • AB4 and PS-F cells were cultivated in 75 cm 2 flasks (Nunc) with RPMI, 10% FCS (PAA), then the cultures were kept in serum-free RPMI (PAA) for 48h. After that AB4 cells were harvested by centrifugation and resuspended in 0.5% (v/v) Triton at a concentration of 2x10 ⁇ 6 cells/ml, PS-F cells were treated with Trypsin (PAA) and resuspended in 0.5% Triton at a concentration of 2x10 ⁇ 6. Culture supernatant of both cell lines was collected and concentrated with U-Tube Concentrator, 2H-2 (Merck).
  • the membrane was incubated with Immun-Star Goat Anti-Rabbit (GAR)-HRP Detection Kit (Bio-Rad Laboratories) and analyzed with a ChemiDoc MP system (Bio-Rad Laboratories).
  • GAR Immun-Star Goat Anti-Rabbit
  • ECIS electrode arrays (8W1E) were obtained from IBIDI, Kunststoff. Before seeding cells, arrays were coated with Fibronectin (Sigma-Aldrich, F1141) at 5 ⁇ g/ml in ultra pure water at 37°C for 3 - 4 hours. After coating, aspirate fibronectin, dry at room temperature and stored under sterile conditions. Depending on cell type and application, different cell concentrations may be used; 1 ⁇ 10 5 cells/ml resulted in a confluent layer within 2-3 days
  • Angiogenesis was measured using ECV304 cells grown in culture vessels coated with fibrinogen/thrombin. Briefly, 30 ⁇ L Fibrinogen Solution was dispensed into wells of a flat-bottomed 96-well plate. The plate was gently shaken to ensure that the fibrinogen solution covers the bottom of the well followed by addition of 20 ⁇ L/well thrombin to the 96-well plate. Plates were shaken and placed at 37°C for 15-60 min for polymerization. The proteins to be tested (r huStat1) were diluted in serum-free medium.
  • ECV 304 cells 10 5 cells/ml
  • serum-free medium 100 ⁇ L of ECV 304 cells (10 5 cells/ml) in serum-free medium were added to wells of 96-well plate and then additional 100 ⁇ l of the respective r hu Stat1 dilutions.
  • hu rVEGF-165 (Peprotech, UK) was used. Cultures were incubated at 37°C, 5% CO 2 , and analyzed by light microscopy/digital photography after 24, 48 and 72hrs.
  • NK+ cells were isolated by negative selection with magnetic beads according to Milteny's protocol. (NK Cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the cell population was determined by flow cytometry. The resulting NK-Fraction was harvested, counted and purity was checked by FACS-analysis.
  • A431 cells were seeded into 8-well ECIS arrays. After 24 hrs, 1 x 10 6 NK cells were added to each well (target:effector ratio 1:10). Recombinant hu Stat1 at different concentrations were added; r hu IL2 (50 units/ml; Peprotech, UK) was used as a positive control. The impedance of the target cell line was observed for several days in order to assess NK cell mediated killing. To one well of A431, no NK were added in order to determine the time-point at which A431 die without induction.
  • SN-F cells primary human skin fibroblasts
  • RPMI-1640 medium supplemented with 10% FCS, 2 mM L-glutamin and Penicillin-Streptomycin. After reaching a confluence of about 80%, cells were trypsinized and seeded on 6-well plates with a cell number of 200 000 cells/well.
  • rhuStat1 at concentrations of 5 ⁇ g/ml, 50 ng/ml and 500 pg/ml were added to cultures. After the end of stimulation time (2h, 6h, 24h, 48h), cells were detached from the wells and further processed for RNA isolation.
  • RNA preparations were further processed for cDNA synthesis with Promega's "ImProm IITM Reverse Transcription System” (Promega, Madison, USA).
  • Promega's "ImProm IITM Reverse Transcription System” Promega, Madison, USA.
  • two different mastermixes were used. Each mastermix was supplemented with Combinatorial Enhancer Solution (CES), which consists of 2.7 M betaine, 6.7 mM DTT (dithiothreitol), 6.7% DMSO (dimethylsulfoxide) und 55 ⁇ g/ml BSA (bovine serum albumine).
  • CES Combinatorial Enhancer Solution
  • Primers were designed with the help of the internet tool Primer Design Assistant (PDA) and synthesized by VBC Oligotech (Vienna, Austria). All primers were used at a final concentration of 10 pmol. Primers are given in table 1: Table 1: Primer sequences (SEQ ID NO 4 to 21 in descending order) Growth factor Primer sequence Size of product (bp) IFN alpha F: CCTGGATAACAGGAGGACCTTG 400 R: CCAGGCACAAGGGCTGTATTTC IFN beta F: CTGCCTCAAGGACAGGATGAAC 350 R: TGACTATGGTCCAGGCACAGTG PDGF-B F: CCCCACACTCCACTCTGATT 181 R: GCCCTGGCCTCTAGTCTTCT FGF-2 F: ATGAAGGAAGATGGAAGATT 216 R: TCAGCTCTTAGCAGACATTG HGF F: CAAATGTCAGCCCTGGAGTTCC 400 R: AATTGCACAGTACTCCCAGCGG TGF beta F: CACGTGGA
  • Agarose gel electrophoresis of the PCR samples was performed in a 1% agarose gel (1 g agarose + 100 ml 1 x Tris borate EDTA (TBE) buffer). Instead of ethidium bromide, the gels were stained with 7 ⁇ l Lonza® Gel Star Nucleic Acid stain. For analysis 15 ⁇ l of the PCR sample and 10 ⁇ l of the DNA ladder (Quick-Load® 100 bp DNA ladder, New England Biolabs) were loaded onto the gel and electrophoresis was run at 115 V for approximately 45 minutes. Pictures of the ready gel was taken under a UV transilluminator.
  • Image analysis was performed with the software LabImage 1D by Kapelan (Leipig, Germany). With the help of this program the amount of DNA was quantified by comparing the DNA bands of the samples with the DNA amount in the marker.
  • TMBP3 Human primary fibroblasts
  • RPMI medium + 10% FCS + TMBP3 unstimulated
  • Lysis Buffer part of array Kit
  • the lysates were rocked at 4°C for 30 minutes. After microcentrifugation for 5 minutes, the supernatant was transferred into a clean test tube. All further steps were done according to R&D's protocol provided with the Proteome ProfilerArray (R&D, Cat. No. ARY012) specific for hu soluble receptors.
  • the light intensity of the signals was measured with the UVP BioSpectrum AC Imaging System (UVP, Upland, CA.) with an exposure time of 30 minutes.
  • the resulting pictures were analysed with the software Phoretix Array, which is part of the TotalLab TL100 program (Nonlinear Dynamics Newcastle, England). This software calculates the pixel value (light intensity) of each individual spots.
  • PET Positron Emission Tomography
  • Stat1 was labeled with iodine-124 and Chloramine T followed by paper electrophoresis and HPLC for quality control. 3 male Spraque Dawley rats were wounded. Rats were anesthetized and shaved on the back of the body. Two full thickness wounds were prepared by the veterinary surgeon using a biopsy-punch (Fa. Henry Schein ® , diameter: 0.8 cm). After wound setting, animals were positioned on the microPET bed which was kept at 38°C. Animals were treated with 5 ⁇ g or 100 ⁇ g r hu Stat1 in 50 ⁇ l hydrogel (Normlgel®, Mölnycke).
  • Purified recombinant Stathmin was diluted in 0,9% (w/v) NaCl, respectively wound fluid and human serum at a concentration of 100 ⁇ g/ml and incubated at 37°C for a total of 72h. Samples (15 ⁇ l) were taken at different time intervals and separated on a 12% Criterion XT Bis-Tris Gel (Biorad). Membrane transfer and blocking was performed as described before. Whole Serum from a rabbit previously immunized with Stathmin was diluted 1:1000 in TBST, applied to the membrane and incubated for 1h at room temperature.
  • the membrane was incubated with Goat polyclonal secondary antibody to Rabbit IgG-H&L-AP (Abcam) diluted 1:1000 in TBST for 1h at room temperature. After another three wash steps membrane was covered with NBT/BCIP stock solution for blot & immunhistochemistry (Roche) diluted according to the supplier's guidelines and incubated at room temperature for 10 min.
  • Alginate-Gel without propylene glycol Alginate-Gel 4% 100.0 g Alginic acid, sodium salt 4.0 g Demineralised water 86.0 g Alginate-Gel 6% 100.0 g Alginic acid, sodium salt 6.0 g Demineralised water 84.0 g
  • Alginate-Gel with propylene glycol Alginate-Gel 4% 100.0 g Alginic acid, sodium salt 4.0 g Propylene glycol 5.0 g Demineralised water 81.0 g Alginate-Gel 6% 100.0 g Alginic acid, sodium salt 6.0 g Propylene glycol 5.0 g Demineralised water 79.0 g
  • a plastic mortar with a pestle has to be tared on an analytic balance.
  • the alginic acid sodium salt has to be weighted into the plastic mortar and an equal amount of demineralized water has to be added. Carefully, the salt will be grinded with this fraction of water. It has to be paid heed to the fact, that the alginic acid sodium salt must not be stuck on the ground of the plastic mortar. To remove it a spatula can be used. Under carefully stirring the remaining water has to be added slowly and partly to the mixture. It has to be paid heed to avoid bubbles in the formulation. After stirring for a while the alginic acid sodium salt will be dispersed in the water and will starting gelling. Now the plastic mortar has to be stored in the refrigerator for at least 12 hours.
  • the produced gels have to be sterilized.
  • the samples will be placed in an autoclave for 30 min at 120 °C.
  • stathmin as therapeutic protein
  • the therapeutic protein has to be solved in water.
  • the drug concentration was decided on 500 ⁇ g/ml.
  • 10 g of a protein solution with the decided concentration has to be filtered through a sterile filter with a pore size of 0.45 ⁇ m.
  • the sterile aqueous solution was injected into the gel. Homogenization was thereafter done by mixing the gel and the protein solution in the vessel in which the gel was sterilized. Each formulation had to be homogenized on the vortex.
  • the formulation was then stored in the refrigerator at 2 °C to 8 °C.
  • Stathmin is secreted by human cells resembling plasmacytoid dendritic cells
  • AB4 cells are permanently growing human cells isolated from a lymph node of a patient with histiocytosis. These cells resemble plasmacytoid dendritic cells since they express various functional and phenotypical features of this particular cell type (i.e. membrane antigen expression, antigen presenting capacity, dendritic shape, high IFN-type 1 expression). AB4 cells were cultured in serum-free medium for 48 hrs and both cell lysates and culture supernatants were analyzed by a sensitive western blotting procedure, which could easily detect Stat1 down to a level of 5 ng. In both lysate and supernatants of AB4 cells a clear cut signal at 17,3 kD could be detected by a Stat1-specific rabbit immune serum.
  • Electric cell-substrate impedance sensing was used to carry out wound healing and cell proliferation in tissue culture.
  • the human epithelial cell line A431 was cultured in ECIS electrode arrays for 48hrs. At that time cells have reached confluency and impedance reached a plateau. Data acquisition was briefly suspended, and wells received an elevated field pulse, one for 10 sec and the other for 30 sec; data acquisition was then restarted. Immediately after the pulse, various concentrations of hu Stat1 were added to individual cultures. 10% FCS was added in positive control wells, while negative control cultures received serum-free medium only. Stathmin1 rapidly induced proliferation of cells in the area covering the electrode in a dose-dependent manner, which was comparable to the positive control cultures. Serum-free medium had no effect ( Fig 4 .).
  • cytokine/growth factor gene expression An important feature of chronic wounds is the absence or downregulation of certain cytokine/growth factor gene expression. This is a major drawback, as these proteins support not only epidermal/dermal cell proliferation and migration but also support functions of dendritic cells, mesenchymal stem cells and tissue remodeling. Results are summarized in table 2. As can be seen, primary human skin fibroblasts could be activated by Stat1 to upregulate cytokines/growth factors that are absent in chronic wounds with the exception of PDGF. Furthermore, gene expression of the genes analyzed are vital for cellular functions of immune cells and stem cells residing in the skin.
  • Fig. 8a and 8b results of proteins (IL-8, TIMP-1,-2) important for wound healing are shown.
  • Il-8 which is an important chemoattractant for PMNs is stimulated 8-fold.
  • Bioactive IL-8 is expressed in wounds and enhances wound healing.
  • TIMPs tissue inhibitor of metalloproteinases
  • MMPs tissue inhibitor of metalloproteinases
  • Stat1 Proteins become rapidly degraded in wound fluid.
  • a prerequisite for the use of Stat1 as a biologic for treatment would be its stability both in wounds as well as formulated in a hydrogel, the formulation planned to be used in clinical trials.
  • two lines of experiments were conducted:
  • the aim of the study was the evaluation of local and system-ical distribution of radiolabelled hu STAT1 after topical administration into a full thickness wound.
  • hu STAT1 treated animals show biologically significant smaller wound areas compared to the PDGF treated animals ( Fig.12 ). Despite that, the healing of the wounds follows a dose dependent manner. Moreover, hu STAT1 accelerates wound healing. The fastest healing of the wounds was recorded in the hu STAT1 high dose group (25 ⁇ g/wound) with statistically significant advantages over the PDGF group on days 9 and 10. Taken together, hu STAT1 accelerates healing in animals with chronic wounds and is superior to current standard treatments.
  • stathmin In combination of stathmin with collagen wound healing was further increased with rapid epithelialization and wound closure ( Fig. 5 ).
  • Stathmin speeds up delayed wound healing in Zucker rats and is not systemically active. In addition, it combines several advantages which are crucial for healing of chronic wounds: promotion of skin cell proliferation, induction of angiogenesis and activation of the natural killer cell activity.
  • recombinant Stat1 is useful for activation of specific cytokine genes and proteins important for immune modulation and cancer therapy.

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